Hydrophobins are a class of small secreted cysteine-rich proteins of fungi or proteins of bacteria that assemble into amphipathic films when confronted with hydrophilic-hydrophobic interfaces. Some hydrophobins form unstable, amphipathic films and others form extremely stable, amphipathic films. By assembling at a cell wall-air interface, some hydrophobins have been shown to provide for a hydrophobic surface which has the ultrastructural appearance of rodlets as on aerial hyphae and spores. Some hydrophobins have been shown to assemble into amphipathic films at interfaces between water and oils, or hydrophobic solids, and may be involved in adherence phenomena. It appears that hydrophobins are among the most abundantly produced proteins of fungi and individual species may contain several genes producing divergent hydrophobins, possibly tailored for specific purposes.
Hydrophobins have been implicated in various developmental processes, such as formation of aerial hyphae, fruit bodies and conidia, and may play essential roles in fungal ecology including spore dissemination, pathogenesis and symbiosis. Hydrophobins fulfill a broad spectrum of functions in fungal growth and development. For instance, hydrophobins are involved in formation of hydrophobic aerial structures (e.g., aerial hyphae and fruiting bodies) and mediate attachment of hyphae to hydrophobic surfaces resulting in morphogenetic signals. The mechanism underlying these functions is based on the property of hydrophobins to self-assemble at hydrophilic-hydrophobic interfaces into amphipathic films.
Hydrophobins secreted by submerged hyphae diffuse in the aqueous environment and may self-assemble at the interface of the medium and the air. This is accompanied by a drop in water surface tension, enabling hyphae to breach the interface and grow into the air. On the other hand, hydrophobins secreted by hyphae that contact a hydrophobic environment will self-assemble at the hyphal surface. The hydrophilic side of the amphipathic film interacts with the hydrophilic polysaccharides of the cell wall, while the hydrophobic side becomes exposed to the hydrophobic environment. Thus, aerial hyphae and spores become hydrophobic, while hyphae that grow over a hydrophobic substrate firmly attach to the hydrophobic substrate. Hydrophobins are, thus, active in the environment of the fungus and at the hyphal surface. Moreover, the hydrophobins also function within the matrix of the cell wall where the hydrophobins somehow influence cell wall composition. In this case, monomeric hydrophobins, rather than self-assembled hydrophobins, seem to be involved.
The best characterized class I hydrophobin is SC3 of Schizophyllum commune, but as far as is known by testing, other members of this class have similar properties. Upon contact with hydrophilic-hydrophobic interfaces, SC3 monomers self-assemble into a 10 nm thick amphipathic film. The hydrophilic and hydrophobic sides of the SC3 membrane have water contact angles of 36° and 110°, making these sides moderately hydrophilic (comparable to carbohydrate) and highly hydrophobic (comparable to TEFLON), respectively.
Interfacial self-assembly of SC3 involves several conformational changes. β-Sheet rich monomers initially adopt a conformation with increased α-helix (α-helix state). SC3 is arrested in this intermediate state at the water-TEFLON interface, but at the water-air interface, the protein proceeds to a form with increased β-Sheet. Initially, this so-called β-Sheet state has no clear ultrastructure (β-Sheet I state), but after a few hours, a mosaic of bundles of 10 nm wide rodlets is observed (β-Sheet II state). This ultrastructural change is not accompanied by a detectable change in secondary structure. The transition from the α-helix state to β-Sheet state can also occur at a water-solid interface, but has to be induced by increasing the temperature and by adding detergent.
Upon self-assembly, the properties of hydrophobins change. Hydrophobins in the β-Sheet state are highly surface active, while monomers have no detectable surface activity. Moreover, lectin activity is increased. In addition, the α-helix state form appears to be less stable than the β-Sheet state. Although both forms strongly adhere to hydrophobic surfaces, the α-helix form can be dissociated and converted to the monomeric formation by treatment with cold, diluted detergents. In contrast, the conformation of the β-sheet form and its interaction with the hydrophobic solid is not affected by this treatment.
Known hydrophobins (see for example WO 96/41882 which also provides guidance to obtain genetically modified hydrophobin-like substances) commonly are proteins with a length of up to 125 amino acids, with a conserved sequence Xn-C-X5-9-C-C-X11-39-C-X8-23-C-X5-9C-C-X6-18-C-Xm (SEQ ID NO: 5) wherein X, represents any amino acid, and n and m independently represent an integer as disclosed by Wessels et al. (ref. 8).
Most classical hydrophobins contain the eight conserved cysteine residues that form four disulphide bridges. However, when the disulphide bridges of a hydrophobin are reduced by chemical modification and the sulfhydryl groups are blocked with, for example, iodoacetamide, the protein assembles in water in the absence of a hydrophilic-hydrophobic interface. The structure is indistinguishable from that of native hydrophobin assembled at the water-air interface. Apparently, the disulphide bridges of hydrophobins keep monomers soluble in water, e.g., within the cell in which they are produced or in the medium, allowing self-assembly at a hydrophilic-hydrophobic interface but are not necessary to provide for its amphiphatic character per se.
Class I and class II hydrophobins are known, each being about 100 amino acids in length and having characteristic hydropathy patterns. Most, but not all, hydrophobins contain eight conserved cysteine residues that form intramolecular disulphide bridges. Hydrophobins may be glycosylated, but the characteristic amphipathic properties of these proteins can be solely attributed to their amino acid sequences. Although the amino acid sequences of class II hydrophobins are relatively well conserved, those of the class I hydrophobins show a low homology. It would be difficult to design universal primers to pick up class I hydrophobin genes by, for example, polymerase chain reaction (PCR).
Indeed, all hydrophobins that have been physically isolated self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. One side of the hydrophobin membrane is moderately to highly hydrophilic (water contact angles below 90°, for example, ranging between 22° and 63°), while the other side exposes a surface with water contact angles essentially above 90°, for example, ranging between 93° and 140°, for example, as hydrophobic as TEFLON (polytetrafluorethylene) or paraffin (water contact angle at about 110°). The contact angle may be determined by a Drop Shape Angle Analysis System, for example, the DSA10MK2 supplied by Krüss.
The membranes formed by class I hydrophobins (e.g., those of SC3 and SC4 of S. commune) are highly insoluble (resisting 2% sodium dodecyl sulphate (SDS) at 100° C.), but can be dissociated by agents such as formic acid (FA) or trifluoroacetic acid (TFA). In contrast, membranes of the class II hydrophobins cerato-ulmin (CU) of Ophiostoma ulmi and cryparin (CRP) of Cryphonectria parasitica readily dissociate in 60% ethanol and in 2% SDS, while assembled CU is also known to dissociate by applying pressure or by cooling.
Self-assembly of hydrophobins is accompanied by conformational changes. Monomeric class I and class II hydrophobins are rich in β-sheet structure. At the water-air interface, class II hydrophobins attain more β-sheet structure (called the β-sheet state), while at the interface between water and a hydrophobic solid, a form with increased α-helix is observed (the α-helical state). The α-helical state seems to be an intermediate of self-assembly, whereas the β-sheet state is likely the stable end-form. At the water-air interface, monomers of class I hydrophobins attain the α-helical state within seconds, but the conversion to the β-sheet state is much slower and takes minutes or even hours. At the water-solid interface, the protein also readily attains the α-helical state, but is thought to be arrested in this intermediate state. The β-sheet end state can be reached by applying a combination of heat and diluted detergent. Both forms of the assembled hydrophobin have an amphipathic nature and can be dissociated with TFA, which unfolds the protein.
After removing the solvent and dissolving in water, class I hydrophobins refold to the same monomeric structure that was observed before purification or TFA treatment. However, self-assembly and disassembly of class II hydrophobins can also be repeated even after dissociation of the membrane by TFA. This shows that both classes of hydrophobins are highly resilient to this type of treatment. The membrane of class I hydrophobins is characterized by a mosaic of bundles of 5-12 nm-wide parallel rodlets. In contrast, rodlets have not been found at surfaces of the assembled class II hydrophobins CFTH1 of Claviceps fusiformis, CRP of C. parasitica, and HFB1 and HFB2 of Trichoderma reesei. Whether the absence of rodlets or the differences in rodlet diameter has any functional significance is not yet known.
The rodlets of the class I hydrophobins, SC3 and SC4, of Schizophylum commune are very similar to the fibrils formed by amyloid proteins. The rodlets of the class I hydrophobins consist of two tracks of 2-3 protofilaments with a diameter of about 2.5 nm each, have a high degree of β-sheet structure, and interact with the fluorescent dyes Thioflavine T (ThT) and Congo Red. Both dyes may be used as probes to discriminate between the α-helical state and the β-sheet state, each having a high propensity for β-sheet state, but no or lower propensity for α-helix state or soluble hydrophobin-like substance.
In addition, SC3 and amyloid proteins self-assemble via intermediates and only above a critical concentration. It was suggested that amyloid fibril formation is common to many, if not all, polypeptide chains. However, because formation of amyloid fibrils is accompanied by loss of function or even disease (e.g. Alzheimer's disease), evolution would have selected against the propensity to form such fibrils. Yet, one or two mutations in a protein suffice to considerably increase the tendency to form amyloid fibrils. To our knowledge, hydrophobins are the first example of functional amyloids with multiple functions in fungal development. Recently, it was found that the four disulfide bridges of the SC3 hydrophobin are essential to prevent the protein from forming the amyloid structures in the absence of a hydrophilic-hydrophobic interface. When the disulphide bridges were reduced and the sulfhydryl groups blocked with iodoacetamide, the protein spontaneously assembled in water. The protein's structure was indistinguishable from that of native SC3 assembled at the water-air interface. Apparently, the disulphide bridges of hydrophobins keep monomers soluble in water (e.g., within the cell or in the medium) and, thus, prevent precocious self-assembly. This would explain why most hydrophobins have eight conserved cysteine residues in nature.
Hydrophobins belong to the most surface-active molecules. With a maximal lowering of the water surface tension from 72 to 24 mJ m−2 at 50 μg ml−1, SC3 is the most surface-active protein known. Other hydrophobins are also highly surface active. Their surface-lowering activities are at least similar to those of traditional biosurfactants. In contrast to these surfactants, surface activity is not dependent on a lipid conjugate, but is solely caused by the amino acid sequence. Moreover, while the maximal lowering of the surface tension by the traditional surfactants is attained within seconds, it takes minutes or even hours in the case of class I hydrophobins. This is explained by the fact that hydrophobins lower the water surface after self-assembly that is accompanied by conformational changes in the molecule.
Despite the fact that hydrophobins have diverged considerably, their gross properties are similar. This flexibility is also illustrated by the fact that removing 25 out of 31 amino acids preceding the first cysteine residue of the SC3 hydrophobin to generate truncated SC3 by genetic engineering only affected the wettability of the hydrophilic side of the assembled hydrophobin. A most remarkable hydrophobin is the trihydrophobin CFTH1 of C. fusiformis. It contains three class II hydrophobin-like units, each preceded by a Gly-Asn-rich repeat and still behaves like other class II hydrophobins. Because of the interfacial self-assembly into amphipathic protein films, hydrophobins can change the wettability of surfaces.
One method to measure wettability is by estimating or measuring the contact angle that a water drop makes with the surface. A large contact angle (>90°) indicates a hydrophobic surface; a small contact angle (<90°) indicates a hydrophilic surface. Furthermore, in gas/liquid or liquid/liquid systems, such as in vigorously shaken water or in oil-in-water or water-in-oil dispersions, air bubbles or oil droplets in solution of hydrophobin become coated with an amphipathic film that stabilizes them.
Solid/liquid interfaces show the same stabilization. For example, a sheet of hydrophobic plastic, such as TEFLON (contact angle 110°), immersed in hydrophobin becomes coated with a strongly adhering protein film that makes the surface completely wettable (contact angle 48°), even after SDS extraction (contact angle 62°), and hydrophobin monomers dried down on a hydrophilic surface make the surface hydrophobic.
The classical hydrophobins are typically isolated from fungi like Schizophyllum commune (ref. 8), but can also be made recombinantly; or comprise a polypeptide having at least 40% identity and at least 5% similarity to at least one polypeptide selected from the group consisting of amino acids 29-131 of SEQ ID NO: 1 and amino acids 29-133 of SEQ ID NO: 2. Such a protein may originate from a filamentous bacterium, in particular, a bacterium capable of forming aerial hyphae, such as an Actinomycete, and, more specifically, the filamentous bacterium may be a Streptomyces species. A Streptomyces species, from which the protein may be isolated using standard procedures for the isolation of hydrophobins, is a Streptomyces species which has been transformed with a construct that can be isolated from an E. coli strain deposited on 14 Mar., 2000 under accession number CBS 102638 with the Centraalbureau voor Schimmelcultures (Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, the Netherlands). This is disclosed in PCT/NL01/00268.
In particular, Wessels et al. (Advances in Microbial Physiology, 38, pp. 1-45 (1997)) suggest attaching small ligands to a layer of hydrophobin via covalent binding or coupling of amino groups to aldehyde groups on mannose residues (Id. at p. 35). The only example given relates to coupling a protein molecule onto a layer of hydrophobin present on a gold surface. However, coating the surface with a hydrophobin may reduce the sensitivity of the sensor as less surface area is available or reactions to be detected take place at a greater distance from the sensor surface.